Using high-dimensional immunophenotyping for mapping immune cell composition in rapid cryopreserved liquid biopsies

Liquid biopsies, such as a blood sample, are non-invasive and simple ways to monitor biomarkers in clinical settings. For cancer immunotherapy settings a major clinical goal is to identify host immunological biomarkers that can be used to monitor progression of disease, treatment efficacy, or even adverse effects. Here, the profiling and validation of blood circulating immune cells may allow continuously survey on how the immune system alters during disease progress and treatment. However, such practice will require proper sampling and development of a robust high-dimensional phenotypic analysis. Here, we designed a 37-marker spectral flow cytometry assay with the purpose of conducting high-dimensional immunophenotyping as monitoring the immune status in patients. The assay was developed to profile all major immune cell compartments, their differentiation as well as activation markers, including numerous checkpoint markers. Importantly, we validated this assay in context of how liquid biopsies should be processed to ensure minimal effect of cryopreservation compared to immunophenotyping of a freshly acquired blood sample. Our work highlights important considerations for which phenotypic alternations can be introduced with different cryopreservation methods. Essentially, we identified a fast and simple processing pipeline where cryopreservation of immune cells showed minimal changes in the immunophenotyping profile. This approach can certainly be implemented in clinical settings and will help diagnostic clinical sites with a strong and validated tool applicable for monitoring individual patients immunological status.